Fluorescent conjugate for differentiating between diseased and healthy tissues

ABSTRACT

The present invention relates to conjugates comprising a compound capable of emitting fluorescence, a linker and a protein. The present invention also concerns the preparation of such conjugates as well as their use.

The present application is a United States national stage pursuant to 35U.S.C. §371 of PCT application PCT/DE97/00166 filed on Jan. 23, 1997.

FIELD OF THE INVENTION

The present invention relates to conjugates for distinguishing unhealthyor pathological tissue from healthy tissue, a process for thepreparation of such conjugates as well as their use.

BACKGROUND OF THE INVENTION

When pathological tissue is treated, its removal is often an essentialstep. For this purpose, it is necessary for the operating surgeon tounderstand clearly where pathological tissue ends and healthy tissuebegins. However, this is often impossible. Therefore, spurs ofpathological tissue are overlooked which then represent the basis fornew formation of the pathological tissue.

Thus, it is an object of the present invention to provide a method ofmaking a distinction between pathological tissue and healthy tissue.

BRIEF DESCRIPTION OF THE INVENTION

According to the invention, this is achieved by the subject matterdefined in the claims.

Therefore, the present invention relates to a conjugate comprising afluorescence-capable compound or a compound capable of emittingfluorescence, a linker and a protein.

The expression “fluorescence-capable compound or a compound capable ofemitting fluorescence” includes compounds of any kind that can bestimulated to emit fluorescence. Examples include fluorescent dyes, e.g.xanthene dyestuffs such as fluorescein, aminofluorescein (AFl),erythrosin, aminoerythrosin (AEros), eosin yellowish and 2-aminoeosinyellowish (AEs) as well as derivatives and analogues thereof. The abovecompounds may also have photodynamic activity. An example thereof isAEros. The photodynamic activity is typically stimulated at a wavelengthranging from 500 to 550 nm (AEros: 533 nm). Porphyrins, chlorins andbacteriochlorins are normally exempted from the above compounds.

A conjugate according to the invention may contain several compoundsthat are capable of emitting fluorescence and may be the same ordifferent from one another.

A protein, such as a particularly native protein that is not consideredexogenous, may be present in a conjugate according to the invention. Theprotein preferably has a molecular weight of up to 100,000 daltons,particularly 30,000 to 100,000 daltons. In especially preferredembodiments, the protein is albumin, particularly human serum albumin(HSA), and transferrin. It is also possible to use protein fragments.Like the proteins, protein fragments may effect a concentration of theconjugate in pathological tissues, particularly in tumor tissue and insuperficial relatively small vessels, e.g. neovascularizations in theregion of the cornea (keratoderma of the eye).

The term “linker” comprises compounds of any kind that are suited tolink the compound capable of emitting fluorescence and the protein.Linkers are preferably not cleaved in the body. Examples of such linkersare cyanuric chloride (Cy) and derivatives thereof.

The components of a conjugate according to the invention may be given aseducts. In the conjugate, they are present in derivatized form.Preferred conjugates according to the invention are shown in FIG. 1.

Conjugates according to the invention may be prepared according toconventional processes by which the compound capable of emittingfluorescence, the linker and the protein are bonded, preferablycovalently, with one another. In this connection, reference is made tothe preparation of the conjugates of Examples 1 to 3 by way of example.

Conjugates according to the invention preferably have an increased halflife in the organism. In addition, conjugates according to the inventionpreferably concentrate in pathological tissues, particularly in tumortissue and in superficial, relatively small vessels, e.g.neovascularizations in the region of the cornea. Compounds capable ofemitting fluorescence are stimulated by light, e.g. a UV lamp, so as tomake visible pathological tissues, whereas healthy tissue in which theconjugates according to the invention do not concentrate are not madevisible. Therefore, it is possible to delimit pathological tissues fromhealthy tissue.

Furthermore, the conjugates according to the invention may be stimulatedto develop a photodynamic activity at a wavelength ranging from 500 to550 nm. Light having this wavelength has only a low penetration depth inthe body. Thus, the conjugates according to the invention are suited inthe best possible manner to treat superficial pathological tissues, e.g.neovascularizations and esophageal tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: depicts the AEs-Cy-HSA, AEros-Cy-HSA and AFl-Cy-HSA conjugatesaccording to the invention.

FIG. 2: depicts the concentration of AFl-Cy-HSA in tumor tissue.

DETAILED DESCRIPTION OF THE INVENTION

The following examples explain the invention. They are only exemplary,and the invention is not intended to be limited thereby.

EXAMPLE 1

Preparation of the AEros-Cy-HSA Conjugate According to the Invention

AEros-Cy-HSA is shown in FIG. 1. 50 mg5([4,6-dichlorotriazine-2-yl]amino)fluorescein (AFI-Cy) was dissolved in5 ml DMSO and reacted to form AEros-Cy by adding a corresponding amountof iodosuccinimide in 2 ml DMSO and 1 ml 0.17 M Bic (sodiumbicarbonate). The reaction was terminated after a few seconds. TheAEros-Cy prepared in this way was slowly added with constant stirring to4 g HSA dissolved in 20 ml of original solution, 20 ml. Bic and 20 mlDMSO. The solution adopted a deep-red color. It remained clear. Afterabout 45 minutes, the protein solution was diluted with 11 of distilledwater and then purified by ultrafiltration (YM 30, Amicon) . Theanalytical purity control was made by means of HPLC. The AEros-Cy-HSAconjugate according to the invention was obtained.

EXAMPLE 2 Preparation of the AES-Cy-HSA Conjugate According to theInvention

AEs-Cy-HSA is shown in FIG. 1.

AEs-Cy-HSA was prepared according to the process described in Example 1.

Bromosuccinimide was used in place of iodosuccinimide.

EXAMPLE 3 Preparation of the AFl-Cy-HSA Conjugate According to theInvention

AFI-Cy-HSA is shown in FIG. 1.

45 mg AFl-Cy was dissolved in 4 ml DMSO and slowly added with constantstirring to 4 g HSA dissolved in 20 ml of original solution, 20 ml Bicand 20 ml DMSO. The solution adopted an intensely yellow color duringthe AFlCy addition. It remained clear. After about 45 minutes, theresulting solution was diluted using 11 of distilled water andsubsequently purified by ultrafiltration (YM 30, Amicon). Purity wascontrolled by means of HPLC. AFl-Cy-HSA was obtained.

EXAMPLE 4 Concentration of AFl-Cy-HSA in a tumor

A rat (240 g) having a Walker 256 carcinosarcoma (tumor weight 4.2% ofthe body weight) was given an intravenous injection of AFl-Cy-HSA ofExample 4, which was labeled radioactively beforehand. The rat waskilled after 24 hours. All organs and the tumor were removed. Theamounts of conjugate disposed in the organs and in the tumor weredetermined by measuring the radioactivity. As shown in FIG. 2, theAFl-Cy-HSA conjugate according to the invention concentrated in thetumor.

What is claimed is:
 1. A conjugate consisting essentially of afluorescent dye, at least one linker and native human serum albumin,wherein the linker is cyanuric chloride, and said conjugate is suitablefor in vivo use in distinguishing unhealthy or pathological tissue fromhealthy tissue.
 2. The conjugate according to claim 1, wherein thefluorescent dye is a xanthene dyestuff.
 3. The conjugate according toclaim 2, wherein the xanthene dyestuff is selected from the groupconsisting of fluorescein, aminofluorescein, erythrosin,aminoerythrosin, eosin yellowish and aminoeosin yellowish.
 4. Theconjugate according to claim 1 or 3, wherein the fluorescent dye hasphotoactivity.
 5. The conjugate according to claim 1 or 3, wherein saidconjugate consists essentially of more than one fluorescent dye.
 6. Amethod for preparing a conjugate according to claim 1 or 3, comprisingthe steps of: covalently binding the fluorescent dye, the linker and thenative human serum albumin.